While many questions remain regarding the role of mammalian small RNA pathways in antiviral defense, it is clear that the presence of conserved antiviral silencing pathways in invertebrate organisms plays an important role in the transmission of mosquito-borne viral diseases. Although the small interfering RNA (siRNA) pathway appears to provide the primary antiviral immune response in invertebrate organisms, several lines of evidence suggest an antiviral role for a somatic piwi-interacting RNA (piRNA) pathway present in disease vector mosquitoes. First, the production of piRNAs from viral RNAs described in various mosquito cell lines and tissues is itself suggestive of an antiviral function. Second, in contrast to the enhanced disease phenotype associated with siRNA-deficient mutant flies infected with viral pathogens, an antiviral immune response directed by virus-derived piRNAs modulates the pathogenicity of alphavirus infections in siRNA-deficient mutant mosquito cell lines. Conversly, knocking down components of the piRNA pathway in mosquito cells has been shown to enhance alphavirus replication. Thus, our research plan is designed to test the hypothesis that a non-canonical piRNA pathway present in the soma of mosquito vectors is acting concurrently with the small interfering RNA (siRNA) pathway to form a coordinated, redundant antiviral defense. A model of hierarchical antiviral immunity in disease vectors, based on previous studies in our laboratory, provides a framework for experiments to define the biogenic pathway of viral piRNA production, dissect the relative contributions of the piRNA and siRNA pathways to antiviral immunity and genome integrity, and evaluate the potential of manipulating endogenous piRNA clusters as a broad-spectrum control strategy.